Antibacterial Activity of Moroccan Plants Extracts Against Clavibacter Michiganensis Subsp

Full Length Research Paper

Antibacterial activity of moroccan plants extracts against Clavibacter michiganensis subsp . michiganensis, the causal agent of tomatoes’ bacterial canker

Talibi I., Amkraz N.*, Askarne L., Msanda F., Saadi B., Boudyach E. H., Boubaker H., Bouizgarne B. and Ait Ben Aoumar A.

Faculté des Sciences, LBVRN ; Laboratoire de Biotechnologie et Valorisation des Ressources Naturelles, BP 8106, cité Dakhla, Agadir, 80 000, Morocco.

Accepted 23 June, 2011

In search for alternative ways of tomatoes’ bacterial canker control, we screened here forty medicinal and aromatic plants (MAP) sampled from 15 families, currently used in southern Moroccan traditional medicine, for their activity against Clavibacter michiganensis subsp . michiganensis the causal agent of this disease. The antibacterial efficacy of powders of these plants was determined in vitro using the agar plate’s methods. Results obtained show that all the forty plants tested inhibited the bacterial growth of this pathogen with inhibition zone diameter ranging from 5 to 50 mm. Determination of the minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) of the most effective plants indicates that there plants gender Rubus, Anvillea and Pistacia have the lower MIC which is equal to 3,125 mg ml -1. The other plants ( Lavandula coronopifolia, Lavandula stoechas, Rosa canina, Cistus monspliensis and Cistus crispus ) had a MIC equal to 6.25 mg ml -1. The MBC for different plants tested are between 6.25 mg ml -1, case of Rubus ulmifolius and 25 mg ml -1. The germination percentage of seeds treated with the most efficient three plants extracts was significantly higher than the untreated. Seeds treated with the extract of L. coronopifolia gave the higher germination rate (98%). The rate germination is 84 to 88% respectively for Cistus monspeliensis and Rubus ulmifolius . In other hand population size of the bacterium in the seeds’ surface was significantly reduced compared to the controls for the three treatments, when tomato seeds were artificially inoculated with the pathogen before being treated with the plants extracts (aqueous extracts).

Key words: Bacterial canker, tomato, medicinal plants, biological control.

INTRODUCTION

The tomato crop is subject to attack by a multitude of and destructive disease in tomato crop both under pathogenic microorganisms and its intensive culture has controlled and field cultures (Utkhede and Koch, 2004). generated and amplified the phytosanitary problems. In The bacterial canker can cause significant damage which addition to mushrooms, viruses and deleterious may go up to the destruction of 100% crop (Gitaitis, 1990; organisms, pathogenic bacteria are important factors that Chang et al., 1992). In Morocco, all tomato production reduce the quality and performance of this culture areas are contaminated by this pathogen, whose (Gartemann et al., 2003). The bacterial canker caused by seriousness varies following regions (Fatmi et al., 1989). Clavibacter michiganensis subsp . michiganensis (Smith) In the Souss-Massa Draa region, it has become the main (Davis et al., 1984), account among the most important cause of the premature death of tomato (Fatmi, 1989). phytosanitary tomato problems. It is a very contagious The seeds and infected transplants are the primary sources of primary inoculums of C. michiganensis subsp . michiganensis (Bryan, 1930; Gleason et al., 1991). Bacteria can survive in the soil (Bryan, 1930; Chang et *Corresponding author. E-mail: [email protected]. al ., 1992; Strider, 1967; Farley, 1971), in crop residues Talibi et al. 4333

and multiple alternative hosts (Fatmi and Schaad, 2002). culture of 72 h on the NBY medium. The optical density of bacterial 8 -1 The spread of the disease is ensured by various suspension was adjusted to 0.46 at 610 nm to have 10 cfu ml manipulations like transplantation (Gitaitis, 1990; Gitaitis (Umesha, 2006). A volume of 0.1 ml of the suspension was speared in the medium surface with sterile glass beads. Cultures et al., 1991), phytosanitary treatments and irrigation prepared were subsequently placed in an incubator at 25°C for 30 systems (Strider, 1969; Chang et al., 1992). The min. Sterilized filter paper discs (Whatmann no 3 and 5 mm in chemical treatments recommended for this disease only diameter) were soaked with 10 l of a 10% concentration of each reduced the population of the pathogen in the surface of plant aqueous extract. Discs were placed in the middle of Petri the infected plants (Hausbeck et al., 2000). Although the plates containing pathogen dried cultures prepared previously. Distilled water was used as negative controls. The plates were control of this bacterial disease continues to be difficult, incubated at 25°C and observed after 48 to 72 h. At the end of the prevention was the first defense line. Given the period, inhibition zones around the paper discs were measured and inefficiency of chemical treatments and their impact on compared to the controls. All tests were conducted in fifteen health and the environment, research and development repetitions arranged as five paper discs in three plates for each of alternative methods are recommended. The biocontrol extract. is a promising way and much research works were made worldwide against bacterial canker agent and various Determination of the MIC and the MBC of the efficient plants plant pathogens and encouraging results were fined (Amkraz et al., 2010; Taqarort et al., 2008; Ameziane et The minimal inhibitory concentrations (MIC) of aqueous plants al., 2007; Basim et al., 2006; Umesha, 2006; Utkhede extracts were determined in liquid medium (Schaad et al ., 1995). One suspension of C. michiganensis subsp . michiganensis (H195 and Koch, 2004; Dimitra et al., 2003; Boudyach et al., 8 -1 2004; Ali-Emmanuel et al., 2002; Boudyach et al., 2001). isolate) in exponential growth phase, adjusted to 10 cfu ml was distributed into sterile hemolytic tubes 3.6 ml per tube. A volume of With a view to develop effective natural treatments, 0.4 ml from the stock solution of the plant extract was added to natural extracted substances from plants figure as each tube. We performed a series of 1/2 dilution. The final privileged axes of the biological control of plant concentrations range between 50 and 3,125 mg l -1. Controls pathogens both in crop treatments and in post harvest consisted in tubes with the pathogen suspension without extract. manages. The objective of this study is to select among The tubes were then incubated at 25°C during 24 h. After incubation, the first tube showing visible growth to the naked eye plants harvested in different regions Morocco those which was retained as corresponding to the MIC for each plant. extracts inhibit the growth of C. michiganensis subsp . The minimal bactericidal concentration (MBC) was determined by michiganensis and to assess their activity in vitro. We are spreading 10 l, taken from the MIC tubes, on the NBY agar. The also looking for a biological alternative to the chemical to percentage of viable germs was estimated comparing with controls eradicate the pathogen from the seeds surface performed with decimal dilutions of the bacterial suspension in considered to be the primary source of infection; in the order to obtain a range suspension of bacterial concentrations between 100 and 0.01%. Each dilution 10 l was spread on NBY aim to have a certified seeds without C. michiganensis medium. Cultures thus made were incubated at 25°C for 72 h. The subsp . michiganensis. MBC that corresponds to the plant does allow extract concentration than 0.01% survival of the initial inoculums, was estimated by comparing the number of colonies appeared to that of the initial MATERIALS AND METHODS inoculums with the tenfold dilution series. The MIC of solvent extracts was determined during the previous assay; it corresponds Plant sampling and preparation of the plants extracts to the lower concentration that inhibited C. michiganensis subsp. michiganensis growth. The prospecting and collection of the medicinal plants were conducted during the years 2006, 2007 and 2008 from several regions of Souss-Massa Draa Valley, Agadir, Morocco. The plants Effectiveness of the aqueous extract in the treatment of the harvested were placed in clean plastic bags and numbered. After tomato infected seeds identification in the laboratory, the samples were dried in the shade and in damp shelter for a week, followed by a drying in an oven at The three best plants which extracts inhibit the growth of C. 35°C for one night. After drying, different parts of the plants were michiganensis subsp . michiganensis were used for treating infected transformed to powder. 20 g of powder were put into suspension in tomato seeds. The treatment of seeds and assessment of their one beaker 250 ml containing 60 ml of distilled water. All was germination capacity were made using the method of Tobias et al. brought to a boil during 5 min. Then, the obtained suspensions (2007) modified. Contaminated tomato seeds were mixed with the were double filtered utilizing the gauze followed by filter paper aqueous extract of each plant at a concentration of 500 mg ml -1 for (Whatmann no 1). The filtrate thus obtained was dried in an oven at one hour. Seeds were subsequently dried and then planted in 60°C to get dry extract. The stock solution of each extract alveolus terrines fulfilled with mixture of sand soil and peat (1: 2; corresponded to a concentration of 0.5 g dry extract in 1 ml of v/v). Each treatment was repeated 17 times and each test was distilled water (Ali-Emmanuel et al., 2002). repeated three times. Controls were carried out under the same conditions with seeds dipped in distilled water. The terrines were placed in an experimental greenhouse with randomized design Screening for the antibacterial activity of the aqueous extracts blocs and were watered once every two days. After two weeks, the percentages of the germinated seeds were determined for each The paper disc diffusion method was used to detect the treatment. antibacterial activity of plants extracts. One suspension of C. The second stage of this experiment was to assess the capacity michiganensis subsp . michiganensis H195 isolate (Boudyach et al., of the extracts to eliminate the bacterium from the surface of the 2004) was prepared in the sterile physiological water from a young infected seeds. Artificial inoculated tomato seeds (cv. Close 27) 4334 J. Med. Plant. Res.

with the pathogen isolate suspension at 10 8cfu as described in Effectiveness of the aqueous extract in the treatment Amkraz et al. (2010) were mixed to plant extracts as described of the tomato infected seeds previously. After getting dried for one night under a stream of sterile air, three samples of 5 seeds by treatment were macerated 3- Two weeks after their sowing, seeds germinate but with separately in 1 ml of phosphate buffer (0.05 MPO 4 , pH 7.4) in a sterile mortar and bacterial density in the wash buffer was some differences related to the kind of treatments to determined by serial dilutions on NBY agar medium. Control was which they are subjected (Figure 2). The result showed performed with untreated seeds. Results were expressed as log cfu -1 that the percentage of germinated seeds treated with the seed . three plant extracts is significantly higher than the

untreated. Seeds treated with extract L. coronopifolia Statistical analyses have the highest rate of germination (98%), while germination rates are 84 and 88% respectively for C. All data were subjected to statistical analysis of variance ANOVA monspliensis and R. ulmifolius. The population size of C. using STATISTICA software, version 6, Stat-Soft, 2001, France. michiganensis subsp. michiganensis in the surface of the Percentage values were subjected to arcsine–square infected seeds decreased significantly after the treatment transformation before analysis of variance. Means separation was performed following the Newman and Keels test at P<0.05. with the plant extracts compared to the untreated (Table 3). The most efficient decrease was observed with L. coronopifolia with 72.32% control. RESULTS

Prospecting and collection of plant material DISCUSSION

Our plants collection consists of 40 samples harvested in In this study we have tested the antibacterial activity of 40 different areas of South Moroccan during years 2006, plants harvested in various Moroccan southern regions 2007 and 2008. Plants harvested belonged to fifteen against C. michiganensis subsp. michiganensis , agent of families represented mainly by the Lamiaceae (22.5%), the tomato bacterial canker. The in vitro effect of aqueous the Asteraceae (12.5%) the Fabaceae (12.5%), and solvent extracts of these plants showed that 90 ℅ Rosaceae (10%), Zygophyllaceae (10%) and the inhibit the growth of C. michiganensis subsp. Cistaceae (5%) (Table 1). michiganensis , but to some different proportions. The genus lavandula was the best gender inhibiting growth of C. michiganensis subsp. michiganensis . This result Evaluation of the antibacterial activity of plant concurred with those of Daferera et al. (2003) who extracts reported that Lavender presents an antibacterial effect against some plants pathogens. The essential oil of Lavandula angustifolia inhibits completely C. The antibacterial efficacy of tested plants was determined -1 in vitro using the agar plate’s methods. Results obtained michiganensis subsp. michiganensis growth 600 mg ml show that among the forty plants tested, the aqueous (Daferera et al. , 2003 ). This activity is mainly due to its extracts of all 40 plants inhibited the bacterial growth of bioactive compounds, particularly the linaloole and this pathogen with inhibition zone diameter ranging from acetate of linalyl (Daferera et al. , 2003). The genus 5 to 50 mm (Table 1 and Figure 1). Rubus showed a significant C. michiganensis subsp. michiganensis growth inhibition. Thiem and Go śli ńska (2004) reported that leaf extracts of Rubus chamaemorus

Determination of the MIC and MBC had an antibacterial activity against some Gram negative bacteria. The Rosa kind showed also a strong C. This test was performed with the best plants that have michiganensis subsp. michiganensis growth inhibition. inhibited the growth of C. michiganensis subsp. Basim and Basim (2004) reported that essential oil of michiganensis . The results obtained were presented in Rosa damascena can be used as a bactericidal for the Table 2. From these results the plants gender Rubus, control of fire blight of the rosaceas caused by Erwinia Anvillea and Pistacia have the lower minimum amylovora. Concerning the kind Cistus , Hannig et al. concentration which is equal to 3.125 mg ml -1. The other (2008) showed that Cistus tea has an antibacterial plants ( Lavandula coronopifolia, Lavandula stoechas, activity. This activity is due to the fact that this species is Rosa canina, Cistus monspeliensis and Cistus crispus ) rich in polyphenoles, who had antibacterial properties. had a MIC equal to 6.25 mg ml -1. The MBC for different Similarly, Bouamama et al. (2006) reported that leaf plants tested were between 6.25 mg ml -1, the case of extracts of Cistus villosus and Cistus monspliensis have Rubus ulmifolius, and 25 mg ml -1 for others; L. antibacterial properties against some Gram positive coronopifolia, L. stoechas, R. canina, A. radiata, Cistus bacteria. Difference observed between samples of the monspliensis germination and growth of some same plant’s species can be explained with the difference phytopathogenic fungi and C. crispus. of the phytochemistry composition of plants samples due Talibi et al. 4335

Table 1. List of the forty plants used during present study with there biologic activity against Clavibacter michiganensis subsp. Mchiganensis .

Label N° Botanical name Family Parts used IZ diameter (cm) * 78; 101; 94 Lavandula coronopifolia Lamiaceae Stem, leaves and flowers 4.88 ± 0.19; 4.00 ± 0.00; 1.62 ± 0.04 7 Rubus ulmifolius Rosaceae Stem and leaves 4.05 ± 0.05 43 Rosa canina Rosaceae Stem and leaves 4.04 ± 0.05 114 Cistus monspeliensis L. Cistaceae Stem, leaves and flowers 3.86 ± 0.05 26; 26’ Pistacia atlantica Anacardiaceae Leaves 3.70 ± 0.17; 1.58 ± 0.10 83 Anvillea radiata Asteraceae Stem, leaves and flowers 3.55 ± 0.07 108; 25 Lavandula stoechas Lamiaceae Stem, leaves and flowers 3.33 ± 0.16; 3.07 ± 0.01 107 Cistus crispus Cistaceae Stem, leaves and flowers 3.20 ± 0.04 3 Lavandula maroccana Lamiaceae Stem and leaves 3.04 ± 0.00 90 Cotula cinerea Asteraceae Stem, leaves and flowers 2.86 ± 0.32 79 Linaria sp. Scrophulariaceae Stem and leaves 2.63 ± 0.00 65 Ighermia pinifolia Asteraceae Stem, leaves and flowers 2.59 ±0.00 63; 30 Thymus satureioides Lamiaceae Stem and leaves 2.33 ± 0.24; 1.64 ± 0.05 81 Hammada scoparia Chenopodiaceae Stem and leaves 2.26 ± 0.03 53 Inula viscosa Asteraceae Stem and leaves 2.15 ± 0.29 89 Artemisia inculta Asteraceae Stem and leaves 2.10 ± 0.13 92 Ruta tuberculata Rutaceae Stem and leaves 2.09 ± 0.10 2 Halimium antiatlanticum Cistaceae Stem and leaves 2.06 ± 0.06 105 Witania adpressa Solanaceae Leaves 2.06 ± 0.08 34 Thymus leptobotrys Lamiaceae Stem, leaves and flowers 1.96 ± 0.17 76 Fagonia harpago Zygophyllaceae Stem, leaves and flowers 1.88 ± 0.08 91 Ononis natrix Fabaceae Stem and leaves 1.80 ± 0.03 71; 104 Zygophyllum gaetulum Zygophyllaceae Stem and leaves 1.73 ± 0.17; 1.25 ± 0.07 106 Cleome africana Capparaceae Stem, leaves and flowers 1.60 ± 0.14 62 Ceratonia siliqua Fabaceae Leaves 1.58 ± 0.01 73 Acacia radiana Fabaceae Leaves 1.28 ± 0.04 93 Fagonia zilloïdes Zygophyllaceae Leaves 1.27 ± 0.08 100 Limoniastrum ifniense Plumbaginaceae Stem and leaves 1.26 ± 0.08 86 Psoralea bituminosa Fabaceae Stem, leaves and flowers 0.90 ± 0.00 68 Asteriscus graveolens Asteraceae Stem, leaves and flowers 0.83 ± 0.19 67 Colocynthis vulgaris L. Cucurbitaceae Leaves 0.82 ± 0.14 19 Sanguisorba minor Rosaceae Stem and leaves 0.68 ± 0.14 99 Reseda alba Resedaceae Stem and leaves 0.50 ± 0.00 98 Coronilla terminalis Fabaceae Stem, leaves and flowers 0.50 ± 0.00

*Inhibition diameter of aqueous extracts on disc diffusion essay. Values are means of fifteen replicate ± σ arranged as five discs in three plates for each extract.

to the location, plant’s age and season of the harvesting. promising in this respect; they have an improve action on Phyto chemical analyze of different extracts (essential oil, the germination capacity of seeds and reduced solvent extracts and aqueous extracts) of these plants significantly the viable pathogen cells from the surface of with standard methods like GC/MS, HPLC and CCM the treated seeds. Furthermore, the amount of the (Data not showed) confirmed this hypothesis. medicinal plant extracts to be used as a seed protectors Our finding concurred with all those reported in the for bacterial canker management will be very low literature; the efficient genders in the inhibition of the comparing to their usage for foliar disease or soil that bacterial canker agent were proved to have antibacterial require much more spraying material (Basim et al., 2006). proprieties both in human and plant diseases. As C. michiganensis subsp. michiganensis is known to be transmitted through seeds of the plants (Gittaitis et al., Conclusions 1993) important applications of the extracts can be seed protectors. The three plants tested here appear to be This study led to showing that the South of Morocco is 4336 J. Med. Plant. Res.

Table 2. The MIC and MBC of plants which extracts have given the best degree of inhibition of the pathogen.

Test plants IZ * (cm) MIC (mg ml -1) MBC (mg ml -1) Lavandula coronopifolia (78) 4.88 ± 0.19 a ** 6.25 12.25 Rubus ulmifolius (7) 4.05 ± 0.0 5b 3.125 6.25 Rosa canina (43) 4.04 ± 0.05 b 6.25 25.00 Lavandula coronopifolia (101) 4.00 ± 0.00 b ND ND Cistus monspeliensis L. (114) 3.86 ± 0.05 bc 6.25 25.00 Pistacia atlantica (26) 3.70 ± 0.17 bcd 3.125 12.55 Anvillea radiata (83) 3.55 ± 0.07 cde 3.125 25.00 Lavandula stoechas (108) 3.33 ± 0.16 def 6.25 12.50 Cistus crispus (107) 3.20 ± 0.04 efg 6.25 25 Lavandula stoechas (25) 3.07 ± 0.01 fg ND ND Lavandula maroccana (3) 3.04 ± 0.00 fg ND ND Cotula cinerea (90) 2.86 ± 0.32 g ND ND

* Inhibition diameter of aqueous extracts on disc diffusion essay. Values are means of fifteen replicate ± σ arranged as five discs in three plates for each extract. **Values followed by different letters are significantly different according to ANOVA analyze with Newman & Keuls test at p<0.05. ND: not determined.

A B C

D E F

G H I

Figure 1. In vitro inhibition of the Clavibacter michiganensis subsp. michiganensis growth with some plants’s extracts. (A) Control, (B) Lavandula coronopifolia first sample, (C) Cistus monspliensis , (D) Lavandula coronopifolia second sample, (E) Rubus ulmifolius , (F) Anvillea radiatta , (G) Lavandula stoecha , (H) Pistacia atlantica , (I) Rosa canina . Talibi et al. 4337

c b 100 a b 90 80 (%) 70 60 50 Germination 40 30 20 10 0 Control Cistus Cistus monspeliensis monspliensis RubusRubus ulmofoliusulmifolius Lavandula coronopifolia

Figure 2. Effect of plants extracts on the germination of tomato seeds. The results are analyzed by the ANOVA followed by Newman and Keuls test at P < 0.05. Different letters indicate significantly different results.

Table 3. Effect of plants extracts on the population size of the pathogen in the surface of tomato seeds.

Control L.c. R.u. R.c. C.m. P.a. A.r. L.s. C.c. Population size of the pathogen 5.6±0.92 a 1.55±0.79 d 2.75±0.23 c 3.98±0.85 b 3.70 ±0.31 b 4.68±0.49 ab 4.83±0.54 ab 4.90±0.85 ab 5.19±0.54 ab (log cfu seed -1)

The results are analyzed by the ANOVA followed by Newman and Keuls test at P < 0.05. Different letters indicate significantly different results. L.c . Lavandulla coronopifolia . R.u. Rubus ulmifolius . R.c . Rosa canina. C.m. Cistus monspliensis . P.a. Pistacia atlantica. A.r. Anvillea radiate. L.s. Lavandula stoechas. C.c. Cistus crispus.

rich in species plant with a non-negligible antibacterial for his English correction. They also express there activity towards C. michiganensis subsp. michiganensis . sincere thanks for the anonymous reviewers for their Indeed, the diameters areas of inhibition obtained on comments to improve the manuscript. solid medium oscillate between 5 and 50 mm. The best results are obtained with plants appurtenants to gender Lavandula, Rubus, Rosa, Cistus, Pistacia and Anvillea . REFERENCES

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